Maggie K. S. Tang

Ginsenoside-Rg1 mediates a hypoxia-independent upregulation of hypoxia-inducible factor-1α to promote angiogenesis

Hypoxia-inducible factor (HIF-1) is the key transcription regulator for multiple angiogenic factors and is an appealing target. Ginsenoside-Rg1, a nontoxic saponin isolated from the rhizome of Panax ginseng, exhibits potent proangiogenic activity and has the potential to be developed as a new angiotherapeutic agent. However, the mechanisms by which Rg1 promotes angiogenesis are not fully understood. Here, we show that Rg1 is an effective stimulator of HIF-1α under normal cellular oxygen conditions in human umbilical vein endothelial cells. HIF-1α steady-state mRNA was not affected by Rg1. Rather, HIF-1α protein synthesis was stimulated by Rg1. This effect was associated with constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt and its effector p70 S6 kinase (p70S6K), but not extracellular-signal regulated kinase 1/2. We further revealed that HIF-1α induction triggered the expression of target genes, including vascular endothelial growth factor (VEGF). The use of small molecule inhibitors LY294002 or rapamycin to inhibit PI3K/Akt and p70S6K activities, respectively, resulted in diminished HIF-1α activation and subsequent VEGF expression. RNA interference-mediated knockdown of HIF-1α suppressed Rg1-induced VEGF synthesis and angiogenic tube formation, confirming that the effect was HIF-1α specific. Similarly, the angiogenic phenotype could be reversed by inhibition of PI3K/Akt and p70S6K. These results define a hypoxia-independent activation of HIF-1α, uncovering a novel mechanism for Rg1 that could play a major role in angiogenesis and vascular remodeling.

Exosomes: Emerging biomarkers and targets for ovarian cancer.

The limitations of current chemotherapies have motivated research in developing new treatments. Growing evidence shows that interaction between tumors and their microenvironment, but not tumor cells per se, is the key factor in tumor progression and therefore of obvious scientific interest and therapeutic value. Exosomes are small (30-100 nm) extracellular vesicles which have emerged as key mediators of intercellular communication between tumor cells and major cell types in the tumor microenvironment such as fibroblasts, endothelial cells, and immune cells as well as noncellular extracellular matrices through paracrine mechanisms. This review is to highlight the emerging role of exosomes in particular types of cancer, such as ovarian cancer, owing to its unique route of metastasis, which is capable of rapidly translating exosome research for clinical applications in diagnosis, prognosis, and potential treatment.

BRCA1 deficiency induces protective autophagy to mitigate stress and provides a mechanism for BRCA1 haploinsufficiency in tumorigenesis

Stress adaptation has profound impacts on malignant progression and response to treatment. BRCA1 is an important modulator of cellular stress, but our understanding of its mechanisms of action remains incomplete. Here we identify autophagy as an essential mechanism protecting BRCA1 deficient cancer cells from metabolic stress and allow their survival, which may underlie its significant cancer-promoting properties. We showed that targeted inhibition of endogenous BRCA1 using small interfering RNA caused significant autophagy in response to serum starvation and endoplasmic reticulum stress, whereas overexpression of BRCA1 did not, confirming that the effect was BRCA1 specific. We demonstrated that Beclin 1 was activated in BRCA1 deficient cells, suggesting involvement of a canonical pathway. Importantly, BRCA1 deficient cells were highly dependent on autophagy for survival, and rapidly underwent cell death upon disruption of autophagy. Notably, this dependence on protective autophagy extended to their tissue of origin, as ovarian surface epithelial cells from women testing positive for BRCA1 mutations, in contrast to those with no mutations, robustly induced autophagy to mitigate the stress and promote their survival. These findings highlight a novel role for BRCA1 in protective autophagy, which may make its essential contribution to tumorigenesis and prognosis.

Abstract 1035: An angiogenic role of E-cadherin-positive exosomes in ovarian cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Angiogenesis, the induction of vasculature, is a vital step for metastasis. Loss of E-cadherin is a well-established marker for tumor metastasis. In ovarian cancer, a highly metastatic tumor that is rapidly lethal, we and others have previously shown the deregulated pattern of E-cadherin and its close association with tumor progression. Although E-cadherin is synthesized as a transmembrane molecule, it can be shed from the cell surface (sE-cad) which accounts for a rapid removal of functional E-cadherin. Here, in addition to ectodomain shedding, we show for the first time that sE-cad can be actively released from ovarian cancer cells in the form of exosomes. These sE-cad-positive exosomes play a critical role in angiogenesis which stimulated the migration, permeability, and tubulogenesis of human umbilical vein endothelial cells in vitro, and promoted functional neovascularization in vivo. In search of the underlying mechanisms by which sE-cad-positive exosomes might regulate angiogenic endothelial cells, which lack E-cadherin, we showed that a novel heterophilic crosstalk between sE-cad and VE-cadherin. Moreover, such angiogenic effect was mediated through activation of the phosphatidylinositol 3-kinase/Akt-beta-catenin signaling cascade. These results uncover a new angiogenic function for sE-cad and a novel paradigm for sE-cad production which may have potential clinical implications (This study was supported by RGC grant HKU781013). Citation Format: Maggie K. S. Tang, Alice Sze Tsai Wong. An angiogenic role of E-cadherin-positive exosomes in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2015-1035

Abstract 3790: Loss of BRCA1 regulates autophagy as a cellular stress response in breast and ovarian cancer cells

Inactivation of cell death is a major step in tumor development. The BRCA1 tumor suppressor, which is frequently mutated in human breast and ovarian cancers, is a critical mediator of cell death. Although the role of BRCA1 gene loss for inhibiting apoptosis is well established, little is known regarding its involvement in other cell survival pathways. Increasing evidence strongly supports that autophagy is a key strategy for cell survival in response to stress. Endoplasmic reticulum (ER) stress and nutrient deprivation are common environmental stresses governing the survival and adaptation of tumor cells in vivo. Here we show that BRCA1 is an important regulator of autophagy, particularly under stress conditions. We found that autophagy could be induced by chemicals that cause ER stress and nutrient deprivation in T47D breast cancer cells and OVCAR-3 ovarian cancer cells with targeted inhibition of endogenous BRCA1 using small interfering RNA. This was characterized by conversion of microtubule-associated protein 1 light chain 3 (LC3) I into LC3 II, punctate green fluorescent protein-labeled LC3 fluorescence, and autophagosomes formation. Treatment of cells with the lysosomal inhibitors, Pepstatin A and E-64d, augmented autophagy, suggesting that autophagosomes were actively produced. In contrast, BRCA1-mediated autophagy that was induced under stress conditions was inhibited by treatment with autophagy inhibitor 3-methyladenine. Similar results were obtained in cells with decreased beclin 1 level by RNA interference. These results reveal a novel link between BRCA1 and autophagy, which may contribute to the survival of BRCA1-deficient cancer cells in their microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3790. doi:10.1158/1538-7445.AM2011-3790

Soluble E-cadherin promotes tumor angiogenesis and localizes to exosome surface

The limitations of current anti-angiogenic therapies necessitate other targets with complimentary mechanisms. Here, we show for the first time that soluble E-cadherin (sE-cad) (an 80-kDa soluble form), which is highly expressed in the malignant ascites of ovarian cancer patients, is a potent inducer of angiogenesis. In addition to ectodomain shedding, we provide further evidence that sE-cad is abundantly released in the form of exosomes. Mechanistically, sE-cad-positive exosomes heterodimerize with VE-cadherin on endothelial cells and transduce a novel sequential activation of β-catenin and NFκB signaling. In vivo and clinical data prove the relevance of sE-cad-positive exosomes for malignant ascites formation and widespread peritoneal dissemination. These data advance our understanding of the molecular regulation of angiogenesis in ovarian cancer and support the therapeutic potential of targeting sE-cad. The exosomal release of sE-cad, which represents a common route for externalization in ovarian cancer, could potentially be biomarkers for diagnosis and prognosis.A soluble form E-cadherin is highly expressed in ovarian cancer. Here, the authors show that soluble E-cadherin is released by ovarian cancer cells packaged in exosomes and promotes tumor angiogenesis through β-catenin and NFkB signaling activation.

New insights into the role of soluble E-cadherin in tumor angiogenesis

A key to successful metastasis is the formation of new vasculature, known as angiogenesis. Therefore, it is of great interest to unravel the underlying molecular mechanisms of tumor angiogenesis. Cadherins are a major class of cell surface receptors. The loss of cadherins, especially E-cadherin, is a well-established marker for tumor metastasis. Loss of E-cadherin is also a defining characteristic of several carcinomas, such as lobular carcinoma of the breast, and de-differentiated endometrioid carcinoma of the endometrium and ovary, which are known to be associated with more aggressive tumor behavior. Although E-cadherin is synthesized as a transmembrane molecule, its extracellular domain can be enzymatically cleaved off and released as a soluble E-cadherin (sE-cad), and this accounts for the loss of E-cadherin function or expression that has been implicated in tumor progression and metastasis. Importantly, sE-cad is present at high levels in the serum and malignant ascites of ovarian carcinoma patients. Nevertheless, little is known about how this essential protein dictates metastasis. Hitherto, many studies have given attention only to the dominant negative role of the loss of E-cadherin in weakening cell-cell adhesion, however, it is not known if sE-cad has biological activity in itself. In addition, the release mechanism of sE-cad has remained elusive. Here we show for the first time that sE-cad is a pivotal regulator of angiogenesis. We further show that exosomes are a novel major platform for the cleavage and release of sE-cad in vitro, in vivo and in patients’ derived samples (Nat Commun, 9: 2270).

Abstract 9: An angiogenic function for E-cadherin in ovarian cancer

The loss of E-cadherin, a cell-cell adhesion molecule, is a well-established marker for metastasis of cancer. Although E-cadherin is synthesized as a transmembrane molecule, it can be cleaved of the ectodomain and released in a soluble form (sE-cad), and this accounts a key mechanism for a rapid reduction of functional E-cadherin at the cell surface. Yet, very little is known about how this important protein dictates the metastasis of these tumors. In ovarian cancer, a highly metastatic tumor that is rapidly lethal, sE-cad is highly expressed in the serum and metastatic ascites of ovarian carcinoma patients. Despite many studies focused on the role of E-cadherin loss in weakening cell-cell adhesion, whether sE-cad has biological activity in itself remain virtually unknown. Here we show for the first time that sE-cad can transduce angiogenic signals. sE-cad was also present in the culture supernatant of ovarian cancer lines. sE-cad of supernatant and a recombinant sE-cad-Fc chimera potently stimulated the migration of, permeability, and tubulogenesis by human umbilical vein endothelial cells in vitro. sE-cad also promoted functional neovascularization in a Matrigel implant model in vivo. These effects could be reversed by neutralizing anti-sE-cad, confirming that the effects were sE-cad specific. In addition, we found that sE-cad bound to VE-cadherin, an effect mediated through the phosphatidylinositol 3-kinase/Akt-β-catenin pathway. These results unravel a new and important piece to the complex biology of tumor angiogenesis and metastasis, and provide insights of novel mechanisms regulating angiogenesis (This study was supported by RGC grant HKU781013). Citation Format: Maggie Kei Shuen Tang, Alice Sze Tsai Wong. An angiogenic function for E-cadherin in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 9. doi:10.1158/1538-7445.AM2014-9

Abstract 1722: c-Met overexpression contributes to the acquired apoptotic resistance of non-adherent ovarian cancer cells through a cross-talk mediated by phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2

Ovarian cancer is the most lethal gynecological cancer mainly due to widespread peritoneal dissemination and malignant ascites. Key to this is the capacity of tumor cells to escape suspension-induced apoptosis (anoikis), which also underlies their resistance to chemotherapy. Here we utilized a non-adherent cell culture model to investigate molecular mechanisms of apoptotic resistance of ovarian cancer cells that may mimic the chemoresistance found in solid tumors. We found that ovarian cancer cells acquired a remarkable resistance to anoikis and apoptosis induced by exposure to clinically relevant doses of two front-line chemotherapeutic drugs cisplatin and paclitaxel when grown in three-dimensional (3D) than monolayer cultures. Inhibition of the hepatocyte growth factor (HGF) receptor c-Met, which is frequently overexpressed in ovarian cancer, by a specific inhibitor or small interfering RNA blocked the acquired anoikis resistance and restored chemosensitivity in 3D, not in 2D cultures. These effects were found to be dependent on both phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular-signal regulated kinase (ERK) 1/2 signaling pathways. Inhibitors of PI3K/Akt abrogated ERK1/2 activation and its associated anoikis resistance in response to HGF, suggesting a signaling relay between these two pathways. Furthermore, we identified, as a mechanism of this cross-talk, a central role of Ras. Interestingly, Ras did not lie upstream of PI3K/Akt, whereas PI3K/Akt signaling to ERK1/2 involved Ras. These findings shed new light on the apoptotic resistance mechanism of non-adherent ovarian cancer ascites cells (This study was supported by Hong Kong Research Grants Council 7599/05M and Committee on Research and Conference Grant HKU200807176203 to A.S.T.W.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1722.